HIV is a complex retrovirus containing a number of genes not commonly found in other retroviruses. We are focusing on the analysis of two of these genes, vpu and vif. Vpu is encoded only by HIV-1 and encodes for a small integral membrane protein. Vpu independently regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. We have previously shown that CD4 degradation involves a direct interaction with Vpu via their cytoplasmic domains. While the precise mechanism of degradation is still unclear, we have evidence that it is an energy-dependent process and requires ph HIV is a complex retrovirus containing a number of genes not commonly found in other retroviruses. osphorylation of Vpu. We have now analyzed the mechanism of Vpu-regulated virus release and found evidence that this function of Vpu is correlated with an ion channel activity of its transmembrane domain. Thus, while CD4 degradation requires sequences located in the cytoplasmic domain of Vpu (the TM domain can in this case be replaced by heterologous membrane anchors), Vpu-mediated virus release is sensitive to changes in the Vpu TM domain. From these findings we conclude that the two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. In a separate line of research we investigated virus release of HIV-2, which lacks a vpu gene. We found that Vpu can enhance HIV-2 virus release but only under conditions where the HIV-2 Env glycoprotein is not expressed. In the presence of the HIV-2 Env product, efficient virus release is observed which was not further enhanced by Vpu. We subsequently found that the HIV-2 Env product encodes a Vpu-like function that regulates, similar to Vpu, virus particle release. Like Vpu, HIV-2 Env is active not only in homologous systems but can regulate virus release of heterologous viruses such as HIV-1 or SIV. We are currently investigating which subdomains of the HIV-2 Env protein are involved in this phenomenon. Vif is a 23 kDa basic protein which has an important function in regulating infectivity of progeny virions. The biochemical mechanism of Vif function is obscure. We analyzed the role of Vif by studying its subcellular distribution by cell fractionation as well as confocal microscopy. We found that a substantial portion of intracellular Vif protein is associated with the cytoskeleton, specifically intermediate filaments. The association of Vif with intermediate filaments is specific and can result in the reorganization of the cytoskeletal network. In addition, we found that Vif is incorporated into virus particles where it associates with the core of the virions. Mutational analysis of Vif suggests that a domain located near the N-terminus of Vif is involved in the interaction with vimentin. The interaction of Vif with vimentin results in the partial or complete reorganization of vimentin and, in extreme cases, culminates in the perinuclear aggregation of vimentin and Vif. Vif-dependent collapse of vimentin is cell-cycle dependent and occurs during late S to G2/M phase. It is unclear at this time if and how the association of Vif with vimentin and the resulting changes in vimentin structure are correlated with the effect of Vif on vimentin. Studies involving Vif mutants are ongoing to investigate this problem.